The present invention relates to the field of neurological dysfunction and, more particularly to a neurological disease characterized by impairment of neuromodulator function, as in Parkinson""s disease.
Parkinson""s disease (PD) is characterized by tremors, hypokinesia, rigidity and abnormal posture as the principal visible symptoms. The tremors in PD are of the resting type, since they occur when the muscles are in a state of relaxation. Its main pathological feature is the degeneration of dopaminergic neurons which have their cell bodies in the substantia nigra and their terminals projecting into the neostriatum. Dopamine is thus significantly depleted in the neostriatum of PD patients. Changes to the substantia nigra and the neostriatal complex are linked to the tremors seen in PD. Compounds that damage the nigrostriatal dopaminergic system and cause hypokinesia, rigidity and tremors have the potential to be used as models for studying PD. Chemical agents such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) damage the nigrostriatal dopaminergic neurons and are widely used to induce symptoms of PD. The effectiveness of these compounds rely on their ability to cause significant damage to the nigrostriatal dopaminergic system. The levels of symptoms are apparently dependent on the degree of nigrostriatal damage, which is somewhat difficult to control. As a result, the symptoms produced by these agents are predominantly rigidity, hypokinesia and poverty of movements and are not always consistent. Tremor, the most conspicuous symptom of PD is not a characteristic feature of the MPTP and 6-OHDA models.
Despite the abundant knowledge regarding the behavioral and pathological features of PD, the actual cause of Parkinson""s disease symptoms and neurodegeneration remains elusive. Although dopamine depletion has been shown to be a common feature, it is unclear whether this is due mainly to the loss of the dopamine producing cells themselves. PD models such as that of MPTP, a synthetic compound, do not explain cases of familial Parkinsonism. The present model, however, is based on S-adenosylmethionine (SAM), an endogenous compound that is widely distributed within body tissues. This implies that environmental and genetic factors that induce aberrant changes in its synthesis and use may result in disease conditions. It has been reported that injecting SAM into the lateral ventricles of rodents caused the principal symptoms observed in PD (Charlton, 1990, Charlton and Crowell, Jr., 1995, Charlton and Way, 1978, Crowell, Jr. et al, 1993). Most importantly, tremors were routine and always in association with hypokinesia, rigidity and abnormal posture. SAM injections also caused dopaminergic system degeneration as depicted by the loss of tyrosine hydroxylase immunoreactivity in the nigrostriatum and forebrain (Charlton, 1997).
Without a known cause, no cure or preventive measures have been derived for PD. Clinical management of PD has often involved the use of L-DOPA (Pfeiffer, 1998), an intermediate in the dopamine biosynthetic pathway. The general consensus is that L-DOPA, which is more accessible to the central nervous system than dopamine, is taken up by dopaminergic neurons and enzymatically converted to the active neurotransmitter, thus replenishing the depleted dopamine. It has been proposed that SAM-dependent methylation of dopamine by catechol-o-methyltransferase (COMT) may also deplete DA. COMT inhibitors are therefore being explored as PD medications (Pfeiffer, 1998).
SAM-dependent methylation of molecules capable of altering dopaminergic transmission is by no means limited to DA and its metabolites. Nucleic acid methylation is believed to influence gene expression (Chiang et al. 1996). Deleterious changes in gene regulation may precipitate neurodegeneration similar to that encountered in PD. Also, since many studies have concentrated on events leading to the synthesis and release of dopamine, defects in such important phenomena as the transmission of signals from the post synaptic receptors following dopamine receptor binding are yet to be explored for possible links to PD.
Following release, dopamine initiates a chain of events by binding to the post synaptic dopamine receptor. The dopamine receptors belong to a class of membrane-spanning receptors that interact with the GTP-binding proteins known as heterotrimeric G-proteins (Gudermann et al, 1996). Multiple forms of the respective monomers exist with varying degrees of amino acid sequence similarity. Of the three subunits, the xcex3-subunit undergoes the post-translational modification with either a C15 (trans,trans-farnesyl) or a C20 (all trans-geranylgeranyl)isoprenyl unit. A consensus amino acid sequence (CaaX, where C is cysteine, a is any aliphatic amino acid and X is the carboxyl terminal amino acid) directs the S-prenylation of a cysteine residue (Sinensky and Lutz, 1992). The terminal tripeptide is proteolytically removed (Hrycyna and Clarke, 1992) thus exposing a C-terminally prenylated protein that is capable of undergoing reversible SAM-dependent methylation (Perez-Sala et al, 1991). The xcex3-subunits, which vary in size from 5 to 7.5 kDa (Cali et al, 1992), exist in a stable heterodimer complex with the xcex2-subunit, the xcex2xcex3-complex only transiently interacting with the xcex1-subunit (Clapham and Neer, 1997). The xcex1-subunit and the xcex2xcex3-complex interact with various cellular effector enzymes and ion channels (Clapham and Neer, 1997). For example, the xcex2xcex3-complex was found to inhibit the activity of Ca2+/calmodulin-stimulated type-I adenylylcyclase but increased that of stimulatory recombinant xcex93xcex1-stimulated type-II adenylyl cyclase (Iniguez-Lluhi et al., 1992). These effects on the enzyme activities were found to be dependent on the C-terminal isoprenylation (Iniguez-Lluhi et al., 1992). The xcex2xcex3-complex also binds to and influences the activity of voltage-dependent calcium channels (De Waard et al., 1997) and binds to immobilized Raf-1 protein kinase with a nanomolar dissociation constant (Pumiglia et al, 1995). It is not clear what role methylation may play in these processes, if any.
Farnesylcysteine (FC) and geranylgeranylcysteine (GGC) analogs that mimic the C-terminal portion of the prenylated G-protein xcex3-subunit influence a variety of cellular processes. For example, farnesylthiosalicylate (FTS) increased intracellular calcium concentrations of differentiated HL60 cells and stimulated superoxide release by HL60 cells and polymorphonuclear leukocytes (Tisch et al., 1996). In neutrophils, FC analogs either initiated or inhibited superoxide-release (Ding et al., 1994). FC analogs inhibited [35S]GTP[S] binding to washed membranes of myeloid-differentiated HL60 cells (Scheer and Gierschik, 1993, 1995) and capacitative calcium entry into cells (Xu et al., 1996). Although these compounds are avid methyl acceptors and/or competitively inhibit the SAM-dependent methylation of prenylated proteins (Perez-Sala et al., 1992), the cellular phenomena outlined above are believed to be independent of methylation (Scheer and Gierschik, 1993, 1995, Ding et al., 1994).
The possibility has not been investigated that other molecules involved with dopamine signaling such as the G-protein xcex3-subunit may indeed be hypermethylated and could account for the observed PD symptoms. Methylation has been exert a strong influence on some physiological phenomena. For example, a strong dependence on methylation for the activation of phosphatidylinositol-specific phospholipase C has been reported (Parish et al., 1995). Methylation inhibitors were found to modulate nutrient-induced insulin secretion from rat islets (Metz et al, 1993), permeabilized HIT-T15 cells (Regazzi et al., 1995) and amylase secretion by pancreatic acini (Capdevila et al., 1997). Strains of Schizosaccharomyces pombe with a defective mam4 gene that encodes the prenylated protein methyl transferase produce non-methylated, inactive M-factor (Imai et al., 1997). Additionally, demethylation of S. cerevisiae a-factor resulted in loss of biological activity (Anderegg et al, 1988).
With the foregoing in mind, the present invention advantageously provides a method of substantially reversing a methylation imbalance and its effects in mammalian neural tissue thereby aiding in restoring neuromodulator function. The method comprises contacting the neural tissue with an effective amount of a prenylcysteine compound or a pharmaceutically acceptable analog thereof. The invention also provides a method for treatment of a mammalian neurological disease characterized by impaired neuromodulator function. The treatment method comprises administering a therapeutically effective amount of a composition including a prenylcysteine compound or an analog thereof in a pharmaceutically acceptable carrier. Also included in the invention is a composition for treating a mammalian neurological disease characterized by impaired neuromodulator function. The composition comprises a therapeutically effective amount of a prenylcysteine compound or a pharmaceutically acceptable analog thereof.
Parkinson""s disease (PD)-like tremors, hypokinesia, rigidity and postural abnormalities were dose-dependently induced in rats by intracerebroventricular (icv) injections of S-adenosylmethionine (SAM). A method for quantifying the tremors was devised. The symptoms appeared within 0.5-2 min, peaked within 10 min while recovery occurred about 90 min after SAM injection. The PD-like symptoms were not significantly affected by the inhibitor of SAM-dependent methylation, S-adenosylhomocysteine (SAH), L-DOPA and N-acetyldopamine. [3H-methyl]SAM reacted with rat nerve cell membranes in a timely, specific and irreversible manner. Icv injection of [3H-methyl]SAM resulted in the methylation of a 5 kDa molecule within the same time as the PD-like symptoms, whose chromatographic mobility on a gel-filtration column was altered by denaturation with 6 M guanidine hydrochloride, suggesting possible association with another molecule. Lyophilized aliquots of the radioactive peak fractions released a volatile radioactive compound on incubation with 1 M sodium hydroxide solution. Since PD involves the loss of dopaminergic function, and dopamine signaling is mediated by receptors that interact with trimeric G-proteins whose xcex3-subunits exist in dimeric complexes with the various xcex2-subunits, range in size between 5 and 7 kDa, are prenylated and undergo reversible C-terminal carboxylmethylation, farnesylcysteine (FC) analogs were synthesized and tested to further understand the mechanism by which SAM-induces the PD-like symptoms. The FC analogs, which are modeled on the isoprenylated C-terminal of the G-protein xcex3-subunit, blocked the tremors, rigidity, abnormal posture and not only reversed the hypokinesia but also caused a significant degree of hyperactivity in the experimental animals. This indicates the specificity of the compounds for alleviating the symptoms, and suggests the need for titrating the compounds to determine an effective therapeutic dose. The FC analogs potentiated the amphetamine-induced rotation of 6-hydroxydomaine-lesioned rats. This effect harmonizes our model with at least one other PD model. It also indicates by increasing the effect of dopamine released by the amphetamine injection that 1) SAM induces PD symptoms by affecting the DA signaling pathway, 2) the SAM effect could be corrected by injecting the animals with FC analogs, and 3) giving an amount that surpassed a dose sufficient to obviate the SAM-induced effects could cause a significant degree of hyperactivity. Since SAM and the prenylated proteins naturally coexist in the brain, it is possible that PD symptoms can be precipitated in some individuals through an aberration in the methylation/demethylation homeostasis. Such an imbalance may occur as a result of environmental and/or genetic factors that take effect gradually with aging. The results of our study indicate that derivatives of isoprenyl compounds (geranyl-, farnesyl- and geranylgeranyl-) substantially reverse the deleterious effects of the methylation/demethylation imbalance in neural tissue.
In the studies developing the present invention, we have reported that SAM induced PD-like symptoms in rats are associated with the in vivo methylation of molecules of about 5 kDa in size, which is around the molecular weight range of the trimeric G-protein xcex3-subunit. Lyophilized aliquots of the chromatographic peak fractions containing the methylated products from brain extracts released volatile radioactive groups on incubation with NaOH, suggesting the possible involvement of carboxylic acid moieties. Furthermore, the SAM-induced PD symptoms were blocked if the SAM injections were preceded by an effective dose of an FC analog. The FC analogs not only blocked the tremors and reversed the hypokinesia, rigidity and postural abnormalities but also caused most of the injected animals to engage in persistent hyperactivity. Furthermore, the FC analogs significantly increased amphetamine-induced rotation of 6-OHDA-lesioned rats, thus suggesting that these compounds could be acting directly on the DA signaling pathway. This effect on the 6-OHDA-lesioned rats also underscores the link between the present SAM-PD animal model and previous PD models.
The method of the invention substantially reverses a methylation/demethylation imbalance in mammalian neural tissue to thereby restore neuromodulator function. The neuromodulator also comprises a neurotransmitter receptor site and, particularly, a receptor site for dopamine. The method is accomplished by contacting the neural tissue with an effective amount of a compound including prenylcysteine or a pharmaceutically acceptable analog thereof. The compound in the method is preferably selected from farnesylcysteine, N-acetylgeranylcysteine, N-acetylfanesylcysteine, N-acetylgeranylgeranylcysteine, farnesyl-2-mercaptoethanesulfonic acid, farnesylthioacetic acid, farnesylmercaptosuccinic acid, farnesylthiolactic acid, and farnesylthiotriazole.
The invention also includes a composition and a method for treatment of a mammalian neurological disease characterized by impaired neuromodulator function, preferably wherein the impairment is characterized by a methylation/demethylation imbalance. The method comprises administering a therapeutically effective amount of a composition including a prenylcysteine compound or an analog thereof in a pharmaceutically acceptable carrier.